1. Field of the Invention
The present invention relates to the field of medical immunology and more particularly to monoclonal antibodies that can recognize platelet membrane glycoproteins and hybridomas for producing the monoclonal antibodies. The present invention also provides anti-platelet compositions containing said monoclonal antibodies and uses thereof in anti-thrombotic therapy.
2. Related Art
Diseases induced or complicated by thrombus formation seriously threaten human health and are one of the most common reasons for human death. Accordingly, prevention of thrombus formation has become a highlight in modern medical research. Platelets, as a major component of thrombi, play a critical role in thrombosis. In normal blood circulation platelets exist in a resting state, but when the blood vessel is damaged and the subendothelial matrix, such as collagen, is exposed, platelets will adhere to the subendothelial matrix of the damaged vessel via direct binding of membrane glycoproteins thereof to plasma adhesive proteins such as von Willebrand factor (vWF). The platelets that adhere to the matrix or are activated by some agonist produced in the process of blood coagulation and tissue injury will change their form, spread their pseudopodia and further release intracellular granular contents. Concurrently, the platelet membrane glycoprotein (GP) IIb-IIIa complex is activated to form a receptor for adhesive molecules, the binding of fibrinogen to which then promotes adhesion and aggregation of platelets and ultimately leads to the formation of a platelet thrombus at the damaged vessel wall (Plow E. F, Ginsberg M. H. Cellular adhesion: GPIIb/IIIa as a prototypic adhesion receptor. Progress in Haemostasis and Thrombosis. 1989; 9: 117-124). Platelet thrombosis plays a key role in the pathogenesis of diseases associated with arterial thrombo-embolism, including coronary thrombosis such as occurs during acute myocardial infarction. Consequently, anti-platelet agents have become important therapy for diseases of arterial thrombo-embolism including coronary heart disease (Becker R. C. Thrombin antagonists and antiplatelet agents. Am J Cardiol 1992; 69: 39E; Ruan C. G. Monoclonal antibodies and thrombotic diseases. Science and Technology at the Frontier in China [Ed. By Chinese Engineering Academy] 2001;Vol 4: 131-145). Drugs currently utilized in clinics include Aspirin, Dipyridamole and Ticlopidine etc., which only function at one of the steps in the process of platelet activation and their curative effects are not as good as those of GPIIb-IIIa antagonists. GPIIb-IIIa antagonists directly inhibit the binding of fibrinogen to the GPIIb-IIIa complex receptor, the final common pathway of platelet aggregation, and then inhibit the formation of a platelet thrombus, and accordingly have become specific and potent anti-platelet agents and exhibit favorable prospects for application in diseases associated with thrombosis. There are three kinds of GPIIb-IIIa antagonists: monoclonal antibodies such as c7E3, synthetic peptides such as Eptifibatide, and non-peptide small molecules such as Tirofiban (Ruan C. G. Monoclonal antibodies and thrombotic diseases. Science and Technology at the Frontier in China [Ed. By Chinese Engineering Academy] 2001;Vol 4: 131-145. Verstraete M. Synthetic inhibitors of platelet GPIIb/IIIa in clinical development. Circulation. 2000; 101(6): 76-80).
The technique of producing monoclonal antibodies (McAb) originated in 1975 and had great effects on fundamental research in biology and medical science, as well as in the diagnosis and therapy of diseases. Along with the extensive application of monoclonal antibodies in clinical therapy, however, it has been found that use of murine monoclonal antibody agents in humans leads to the appearance of human anti-mouse antibodies (HAMA); the molecular weight of the antibody is so great that it makes the amount of antibody available in the target site inadequate; which leads to low efficacy of the antibodies per se, and makes the curative effects unsatisfactory. To overcome these defects, humanization of the antibody is carried out, such processes have been developed from studies on preparation of human-mouse chimeric antibodies, preparation of antibody fragments of small molecules such as single chain antibodies and preparation of bifunctional antibodies. For example, 7E3, a murine monoclonal antibody against GPIIb-IIIa, was prepared by American researchers in 1983. (Coller B S, Peerschke E I, Scudder L E et al. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoprotein IIb/IIIa. Journal of Clinical Investigation. 1983; 72(1): 325-328). In 1992, it was reconstructed as the Fab fragment of the human-mouse chimeric antibody named c7E3. (Knight D M, Wagner C, Jordan R, et al. The immunogenicity of the 7E3 murine Fab monoclonal-antibody fragment variable region is dramatically reduced in humans by substitution of human for murine constant regions. Molecular Immunology. 1995; 32 (16): 1271-1281). Large-scale clinical investigation has uncovered the fact that c7E3 has strong anti-thrombotic effects. The application of this Fab fragment in clinic practice has been approved by the FDA. Nowadays it is widely used in the treatment of ischemic heart disease and has shown that there is a favorable future for clinical application of these kinds of drugs.
However, a deficiency of c7E3 is in that it only acts on one epitope on the platelet membrane and blocks the function of the receptor in a single way and thus cannot achieve complete inhibition of platelet aggregation. So it is desirable to develop a novel monoclonal antibody, which can inhibit platelet aggregation more efficiently and effectively.